1. Field of the Invention
The present invention relates generally to the fields of microbiology and immunology. More particularly, it concerns the response of macrophages to endotoxin exposure from Gram-negative bacteria and methods of detecting and treating individuals at high risk for infection by Gram-negative bacteria and inhibiting sepsis and septic shock.
2. Description of Related Art
“Innate” or “natural” immunity is largely subserved by macrophages and NK cells. These cells lack the huge repertoire of pathogen receptors that comprise the afferent limb of “specific” immunity, e.g., the receptors found on T-cells and B-cells. Rather, they rely upon very broadly effective mechanisms for the recognition of invasive organisms. An important case in point concerns Gram-negative bacteria, all of which bear endotoxin (lipopolysaccharide, LPS) molecules in their outer membrane, which trigger a strong immune response on the part of the host which produces a shock-like syndrome, characterized by low blood pressure and hyporeactivity to vasoconstricter agents.
When macrophages are exposed to pure preparations of endotoxin, they secrete numerous cytokine mediators, including tumor necrosis factor (TNF, TNFα), interleukin-1 (IL-1), interferon-α/β, GM/CSF, IL-8 and ultimately smaller “autocoid” molecules, all of which mediate an intense inflammatory reaction. Endotoxin recognition acts as an early warning signal through which a host may mount a timely defense against invasion by Gram-negative organisms. However, widespread activation of macrophages by endotoxin results in the development of septic shock. By most estimates, Gram-negative septic shock is responsible for 100,000 deaths per year in the United States alone. The entire syndrome of hypotension, coagulopathy, pulmonary edema and acute renal failure results, in large part, from the release TNF and other cytokines in response to exposure to endotoxin.
TNF is probably the most important mediator of local inflammatory processes as well as septic endotoxin shock. It is produced quickly and in large amounts by macrophages that encounter endotoxin (Beutler et al., 1985a). TNF causes shock when administered to animals (Tracey et al., 1986), and blockade of TNF synthesis or activity markedly attenuates the lethal effect of endotoxin (Beutler et al., 1985b). The relationship between endotoxin and TNF is therefore directly correlated. Surprisingly, however, little is known about the mechanism by which endotoxin triggers host cells to release TNF.
The cognate receptor for endotoxin, complexed with lipopolysaccharide binding protein (LBP) in plasma, is the GPI-linked cell surface membrane protein CD14. CD14 effectively concentrates endotoxin onto the surface of macrophages and other defensive cells of the host (Wright et al., 1990). However, it does not actually signal the presence of endotoxin, as CD14 has no cytoplasmic component with which to do so.
Endotoxin is known to trigger both tyrosine and serine phosphorylation events within the macrophage cell, and at least in part, ras, raf, MEK, and members of the MAP kinase family are also involved in signal transduction (Geppert et al., 1994). The endpoints of endotoxin signaling include activation of the transcription of TNF and various genes, and activation of the translation of TNF mRNA (Beutler et al., 1986; Han et al., 1990). At the protein level, this stimulation by endotoxin leads to a several thousand-fold augmentation of cytokine biosynthesis by a macrophage cell. But the initial controlling element and event in the signaling pathway of macrophage response to endotoxin has not been identified. Thus, in spite of its importance, most of the endotoxin signaling pathway remains relatively unknown. Recently however, the Toll-like receptor 2 (TLR2) has been suggested to partially mediate lipopolysaccharide-induced cellular signaling (Gerard, 1998; Yang et al., 1998).
Thirty years ago, mice of the C3H/HeJ strain were noted to be specifically and globally unresponsive to endotoxin, while closely related animals of the C3H/HeN or C3H/OuJ substrains exhibited normal responses (Sultzer, 1968). The median lethal dose of endotoxin is more than 100-fold higher in C3H/HeJ mice than in either of these other strains. Macrophages of C3H/HeJ mice fail to produce cytokines in response to endotoxin, and B-lymphocytes of C3H/HeJ mice are not driven to proliferate by endotoxin. While C3H/HeJ mice are highly resistant to the lethal effect of endotoxin, they are unusually sensitive to infection by gram-negative organisms. The mean lethal inoculum with Salmonella typhimurium, for example, is two organisms in C3H/HeJ mice, whereas several thousand organisms are required to kill mice of the C3H/HeN strain. Hence, the ability to sense the presence of endotoxin is required for defense against gram-negative organisms and it is speculated that individuals that suffer from sepsis and septic shock have a similar genetic mutation which causes them to be more susceptible to infection.
These defective responses by the C3H/HeJ mice are the result of a single, codominant mutation, which maps to a position between the widely separated Mup-1 (Major urinary protein) and Ps (polysyndactyly) loci on mouse chromosome IV (Watson et al., 1978). Mice homozygous for the mutant allele of the “Lps gene” are unresponsive to endotoxin, whereas homozygotes for the common allele are normally responsive, whether lethality or cell-based assays are employed as an index. Heterozygotes exhibit intermediate levels of response. The protein encoded by this mutant gene is the most important known determinant of endotoxin-induced TNF biosynthesis, and indeed, of all reactions to endotoxin.
Many attempts to identify the product of the Lps gene, or to clone it have been made. With the recognition that CD14 serves as the principle cell-surface receptor for endotoxin, it was proposed that the Lps gene might encode an associated polypeptide chain with signal transducing potential, or more broadly, an early component of the signal transduction apparatus. Attempts to identify a CD14 binding molecule, which might be the product of the Lps gene, have been pursued by several investigators. Two-hybrid screening, affinity chromatography, and cross linking approaches have thus far each failed to pinpoint a protein that specifically engages CD14. Expression cloning strategies have also been applied in the search for the Lps gene product without success.
Given the occurrence of gram-negative bacteremia and the high and rising incidence of gram-negative nosocomial infections, a certain subset of the population appears to be at high risk to develop endotoxic shock even if adequate antimicrobial therapy is instituted. It would be useful to know which patients are at high risk for gram-negative bacterial infections and sepsis in advance of its onset. Diagnostic methods that predict the risk of infection as well as the clinical course of sepsis could be reasonably applied to most hospitalized patients. In addition, it is clear that there is an immediate and increasing need for new drugs and treatment methods that regulate macrophage response to gram-negative bacterial infections. Unfortunately, given the current lack of understanding of the regulation of the macrophage response to endotoxin, these drugs and methods have not been developed, and patients continue to be at risk for these life-threatening infections.